Design and Synthesis of 4-flurophthalimides as potential anticonvulsant agents

Anticonvulsant activity of phthalimide was discovered in 2000 by molecular hybridization of thalidomide and ameltolide. In our present research we report some new 4-substituted derivatives of phthalimide with good activity against the tonic and clonic seizures. A series of novel 4-flurophthalimides designed using bioisosteric replacement were synthesized by condensation of 4-flurophthalic anhydride with appropriate arylamines. The purity of these compounds was determined by TLC and the chemical structures were confirmed by IR and 1H-NMR spectroscopy. Anticonvulsant activity of prepared compounds was evaluated using MES and PTZ models. Some of the designed compounds significantly protected mice against the PTZ-induced seizure among which, compound 10 with lipophilic and flexible aromatic moiety was more potent than the reference drug phenytoin and was the most potent in this series of phthalimide derivatives. In the MES model, the prepared phthalimide did not show efficient activity. The prepared compounds are active in clonic seizure.


Experimental
Chemical synthesis A group of N-aryl derivatives of the 4-flurophthalimide (compounds 2-13) were synthesized by condensation of the respective aromatic amine with 4-flurophthalic anhydride in acetic acid at reflux temperature (Scheme 1-A). Nitro moiety of compounds 2-5 was reduced to amine using Pd/C and cyclohexene in 2-propanol at reflux temperature (Scheme 1-B) (24-26). All the solvents and chemicals were obtained from Merck and Sigma-Aldrich Company. Thin layer chromatography was performed on plastic TLC plates and TLC spots were visualized using ultraviolet lamp and iodine tank. Melting points were determined using Electro thermal IA 9300 capillary melting-point apparatus (Ontario, Canada). 1 H-NMR and FT-IR spectra were prepared with the Bruker FT-250 and Nicolet 550-FT spectrometers respectively. A Perkin-Elmer model 240-C apparatus was used for elemental analysis and results were considered in range of + 0.4% of the calculated amounts.
Using a procedure similar to that of 2, 4-flurophthalic anhydride and 2-aminobenzonitrile provide the title compound after 24 h that the crude and impure product was recrystallized from ethanol to afford the desired compound as a pale yellow crystals
Using a procedure similar to that of 2, 4-flurophthalic anhydride and 1-naphthylamine provided the title compound after 72 h that the crude and impure product was recrystallized from ethanol to afford the desired compound

Pharmacology
The test compounds were evaluated for anticonvulsant activities using maximal electroshock-(MES) and pentylenetetrazole-(PTZ) induced seizure. Male NMRI mice (20 -25 g; purchased from Pasture Institute, Tehran, Iran) were used for MES test. For PTZ-induced seizure, male wistar rats (150-200 g) were used. Animals were purchased from Pasture Institute (Tehran, Iran) and were kept in controlled light and temperature condition (12 h/12 h light/dark cycle; 22-25 ºC) with free access to food and tap water. The test compounds were dissolved in DMSO and injected intraperitoneally at the dose of 40 mg/kg 30 min before seizure tests. The different test compound-treated groups were compared with the control group (which received DMSO) or phenytoin-treated group (40 mg/kg) as reference drug. The volume of injected drugs/vehicle was 10 mL/kg in mice and 1 mL/kg in rats. Anticonvulsant evaluation by MES test was performed as described previously (33). Electroshock was induced by applying an alternating current (intensity 40 mA, pulse duration 0.2 s, frequency 50 Hz) through ear clip electrodes by a stimulator (Borj Sanat, Iran). The end-point for seizure occurrence was the observation of hind-limb tonic extension (HLTE) in mice. In PTZinduced seizure, the occurrence of stage 5 of Racine score (rearing and falling with forelimb clonus) (34) was considered as the endpoint and the antiseizure effect was evaluated by measuring the number (ratio) of seizured animals out of total number of animals in each group.

Statistical Analysis
The results of PTZ and MES test are presented as seizured/total and protected/ total, respectively and the percent protection against seizure between the groups was analyzed by Chi-square test. P values less than 0.05 were considered as statistically significant.

Chemistry:
A series of novel 2, 5-disubstituted phthalimides were synthesized in good yield according to the method that was mentioned previously (24-26). Condensation of 4-flurophthalic anhydride with appropriate arylamines in glacial acetic acid resulted in desired 2, 5-disubstituted phthalimide. Reduction of nitro group to amine was done using Pd/C and cyclohexene as catalyzer and hydrogen donor respectively (25). The purity of these compounds was determined by TLC and the chemical structures were confirmed by IR and 1 H-NMR spectroscopy.
Existence of absorption bands of amide, nitro, aromatic amine groups in IR spectra approved preparing of our desired compounds. In H-NMR spectroscopy, the proton on the phthalic ring in nitro and amino derivatives are shifted downfield and upfield respectively as compared with 5-unsubstituted compounds.

Pharmacology
The ability of the compounds 2-17 to protect rats against PTZ-and MES-induced seizure was evaluated and the results were presented in Tables 1 and 2, respectively. For both MES and PTZ model, the test compounds were evaluated for anticonvulsant activity at the dose of 40 mg/ kg and were compared with vehicle (control) and phenytoin (40 mg/kg; as reference drug). As shown in Table 1, compounds 2, 3, 5, 7, 8, 9, 10, 13, 14, 16, 17, and also phenytoin significantly protected rats against PTZ-induce clonic seizure compared with the control group. Compound 10 (seizured/total: 0/10) with lipophilic diphenylmethane moiety was more potent than the reference drug phenytoin (seizured/total: 2/10) and was the most potent in this series of phthalimide derivatives. Due to presence of a lipophilic and flexible phenyl group in this ligand, it is suggested that hydrophobic and charge transfer interactions may be involved in drug-receptor profile. Comparing nitro and amine containing compounds revealed that reduction of nitro to amine moiety in compounds 2 and 5 did not affect the potency. Instead, in compounds 3 and 4, this change in chemical structure decreased and increased drug potency, respectively. Evaluation of the activity of compounds 5, 7, and 8 indicated that the existence of electron withdrawing groups (NO 2 , CN and Cl, respectively) in the phenyl ring increased the potency. Comparison of compounds 9, 10, 11, 12, and 13 which contain two aromatic rings in two different position of phthalimide showed that compound 10 with more lipophilic and routable aromatic ring is more active than others.
Results of MES test (as shown in Table 2) indicated that compounds 4, 7, 8, 13, and 17 were ineffective in this model and compounds 2, 3, 14, and 16 showed moderate antiseizure activity. Although MES results showed a trend of attenuating seizure intensity in mice but none of the tested compounds at the dose of 40mg/kg produced significant protection against seizure compared with the control group.

Conclusion
Sixteen analogs of 4-flurophthalimid were synthesized, purified, and characterized by thin layer chromatography, IR, elemental analysis and H-NMR. Ability of prepared compounds to protect against PTZ induced seizure and MES were evaluated in-vivo in mice. Pharmacological results indicated that all the prepared compounds were active in clonic seizure but in the tonic model did not show any efficient activity.
-4flurophthalimides as potential anticonvulsant agents